OBJECTIVE To assess the accuracy of the indentation method for stiffness measurements and to estimate the Young's modulus of the vocal fold using this technique. METHODS Basic science. METHODS Indentation tests were performed using a range of indenter diameters and indentation depths on single- and double-layer silicone rubber models with various cover-layer thicknesses with known geometry and Young's moduli. Measurements were repeated on intact vocal folds and isolated muscle and cover-layer samples from three cadaveric human larynges. RESULTS Indentation on single-layer rubber models yielded Young's moduli with acceptable accuracy when the indentation depth was equal to or smaller than the indenter diameter, and both were smaller than the physical dimensions of the material sample. On two-layer models, the stiffness estimation was similarly influenced by indenter diameter and indentation depth, and acceptable accuracy was reached when indentation depth was much smaller than the height of the top cover layer. Measurements on midmembranous vocal fold tissue revealed location-dependent Young's moduli (in kPa) as follows: intact hemilarynx, 8.6 (range=5.3-13.1); isolated inferior medial surface cover, 7.5 (range=7-7.9); isolated medial surface cover, 4.8 (range=3.9-5.7); isolated superior surface cover, 2.9 (range=2.7-3.2); and isolated thyroarytenoid muscle, 2.0 (range=1.3-2.7). CONCLUSIONS Indenter diameter, indentation depth, and material thickness are important parameters in the measurement of vocal fold stiffness using the indentation technique. Measurements on human larynges showed location-dependent differences in stiffness. The stiffness of the vocal folds was also found to be higher when the vocal fold structure was still attached to the laryngeal framework compared with that when the vocal fold was separated from the framework.